The broad goal of this project is to identify the molecular mechanisms that underlie ion permeation and channel gating and its regulation in inositol trisphosphate receptors (InsP3R). The InsP3R is a ubiquitous family of intracellular Ca2+ release channels that participates in the generation of complex Ca2+ signals that regulate many physiological processes, and that has been implicated in several human diseases. Nevertheless, the molecular mechanisms in the channel that regulate its ion permeation properties, gating (opening and closing) and regulation are poorly understood. Its intracellular location has generally limited studies of its single channel properties, and its ubiquitous expression has impeded the development of robust systems for recombinant mammalian isoform expression. A challenge is to understand the function and regulation of this family of ion channels in order to understand its roles in Ca2+ signaling in normal and disease states. We previously developed patch clamp electrophysiology of isolated nuclei as a powerful approach for studying the single channel properties of the InsP3R in its native endoplasmic reticulum (ER) membrane. During previous funding cycles, we have made considerable progress in understanding the molecular physiology of recombinant mammalian InsP3Rs. We have developed a robust system that now greatly facilitates electrophysiological characterization of recombinant InsP3R channels in a completely null background. Importantly, we developed novel molecular models that can account for diverse regulation of channel gating that we have previously identified. These models provide an invaluable framework for interpreting the molecular consequences of mutations introduced into the channel sequences. Our development of nuclear patch electrophysiology and novel cell systems for mammalian InsP3R expression now enables detailed studies of wild type and mutant channels in native ER membranes. By comparison with nearly all other ion channels, and despite its role in generating diverse Ca2+ signals in nearly all cells that are responsible for regulating numerous cell physiological processes, the molecular physiology of the InsP3R is fundamentally understudied. Furthermore, the InsP3R has been linked to human disease processes, including apoptosis, neurodegeneration, cerebellar ataxias, and cardiovascular disease, and it may represent a therapeutic target in others. Thus, there are compelling reasons to understand the detailed properties of this ion channel. This knowledge could also be important to the development of therapeutic agents that may target InsP3R. Thus, this project could have a major impact. We propose three specific aims to characterize the molecular mechanisms that contribute to permeation, gating and channel regulation. The results of these studies should provide new insights into the molecular physiology of the family of InsP3R Ca2+ release channels.